The expression of estrogen receptor-alpha (ERa) is critical for establishing the hormone responsive phenotype of breast cancers. Functional promoter analysis and hypersensitive site mapping identified the AP2y transcription factor as a regulator of ERa expression and confirmed the location of AP2 regulatory sites in the human ERa gene promoter. Eliminating AP2y expression in ERa-positive cells using siRNA significantly down-regulates the expression of ERa and ERa-target genes. We conclude that AP2y is a key regulator of ERa expression in hormone responsive breast cancer cells. Epigenetic alterations involving methylation and histone acetylation have also been shown to contribute to the control of ERa gene expression. We hypothesize that epigenetic changes involving the AP2 regulatory element may provide an additional mechanism to regulate expression from the ERa promoter. Furthermore, we have identified an interaction between AP2 factors and the tumor suppressor protein p53 and there is substantial evidence showing that overexpression of p53 is capable of inhibiting the transcriptional activity of AP2. Hence, the interaction between AP2 factors and p53 represents another potential mechanism regulating the expression of ERa and may explain the finding that breast cancers with p53 overexpression are usually ERa-negative. Herein we propose to define the mechanisms regulating AP2-mediated activation of ERa and to determine the physiologic processes of hormone response controlled by AP2y. To accomplish this goal we propose to analyze the parameters of hormone response controlled by AP2y in MCF7 cells. We will define the functional domains of AP2 factors that are necessary for regulation of ERa. Using cell models of ERa repression and reactivation, we will determine if epigenetic alterations of the AP2 regulatory region contribute to control of ERa expression. We will investigate the mechanisms of p53-mediated repression of ERa by determining if p53 alters AP2y activity or chromatin interaction. The role of p53 in regulating ERa will be investigated by examining p53 alleles recovered from breast cancers for mutationsthat disrupt the AP2-interaction domain. We will define residues that are critical for the interaction between AP2 and p53 and generate antagonists as a prelude to developing a specific inhibitor of the AP2-p53 interaction. In the future it may be possible to develop a novel therapeutic to induce hormone response in breast cancers by specifically targeting the AP2 pathway.
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